Feature Articles

Injecting New Life into Cell-Based Assays

Noninvasive Analysis of Live Cells

Being able to noninvasively monitor the same live cells in culture over extended time periods could have important implications for monitoring of cell phenotype and drug screening, suggests Molly M. Stevens, Ph.D., professor of biomedical materials and regenerative medicine at Imperial College London.

“Stem cell research and regenerative medicine rely on understanding and manipulating cells that can differentiate and interact with their environment. Primary stem progenitor and lineage-specific cells are the gold standards.”

However, Dr. Stevens also notes that these can be unreliable due to their variable sources or loss of phenotype when maintained in long-term culture. Raman spectroscopy may help overcome these limitations. “Raman microspectrometry is a way to noninvasively characterize live cells. The Raman spectrum provides an intrinsic, global biochemical fingerprint with molecular-level data on cells.”

Raman spectroscopy is a laser-based analytical optical technique that measures photon scattering from chemical bond vibrations. The energy differences allow one to identify and characterize specific chemical bonds present. The Stevens group recently reported in Nature Materials on the use of the technique to distinguish how different stem cells can form bone tissue with a different chemical make-up.

According to Dr. Stevens, Raman spectroscopy can yield some important advantages over conventional cytochemical methods because it provides a rapid, noninvasive means to analyze cells without the need for fixatives or labels.

“An additional advantage is that, while most biological assays characterize only one marker, Raman spectroscopy can provide a cell-specific biochemical signature. We interpret the signatures with the help of our own sophisticated multivariate statistical analyses and can then detect subtle yet reliable changes in cell phenotype. Applications include toxicity testing of drugs, identification of cancerous cells, and as a tool to characterize cellular processes.”

Automating 3-D Construction

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TAP Biosystems’ RAFT™ (Real Architecture for 3D Tissue™) system is used for the generation of consistent multicellular 3-D tissue models.

Increasing numbers of scientists are turning to 3-D substrates on which cells can be grown. Such substrates more closely mimic the natural in vivo physiological state. However, building 3-D tissues can be a slow and inconsistent process. Automating such construction to enhance throughput faces several challenges.

“Ideally, tissues should be created in a quick, consistent, and simple way. This hasn’t been practical before,” reports Rosemary Drake, Ph.D., CSO, TAP Biosystems.

“It’s been a costly and inconvenient process with poor reproducibility. We addressed these issues in collaboration with leading tissue-engineering academic scientists and developed the RAFT system (Real Architecture For 3-D Tissue). This includes a workstation, consumables, and reagents for making a range of multicellular 3-D tissue models.”

The process can take less than an hour. “Extracellular matrix is largely composed of collagen. In the RAFT system, we mix collagen with cells (such as from epithelium, endothelium, nerve, smooth muscle, tendon, and bone) in a 12-, 24-, or 96-well format to form a cell-seeded hydrogel.

“Next, absorbent plungers simultaneously apply gentle compression and absorb some of the liquid from the gel. This results in a 50–100 fold increase in the concentration of the cells and collagen, giving a consistent transparent tissue model in the bottom of the well. During culture, matrix-rich tissue is created.”

Dr. Drake said that “the density of the collagen matrix is the closest we can get to a tissue-like environment, and cells respond to this in a similar manner to cells in vivo. Therefore, these tissue models have broad applicability in cell-based screening, target validation, lead optimization, and toxicity testing.”

She cited a practical example of the technology. “Our academic collaborators seeded human limbal epithelial stem cells onto a layer of fibroblasts in compressed collagen. After three weeks of culture, the cells formed tissue strikingly similar to the human central cornea.”

Although not all assays need to be done in 3-D, Dr. Drake explained that many applications would benefit from such an approach.

“In particular, it would be useful in improving our understanding of how cancer cells invade and move through tissues. Automating such processes provides a consistent way to interrogate more complex cellular processes.”

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